The human immunodeficiency virus (HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders. (see, e.g., Barre-Sinoussi, et al., (1983) Science 220:868-871; Gallo et al. (1984) Science 224:500-503; Levy et al., (1984) Science 225:840-842; Siegal et al., (1981) N. Engl. J. Med. 305:1439-1444; Guyader et al., (1987) Nature 326:662-669).
The envelope protein of HIV-1, HIV-2 and SIV is a glycoprotein of about 160 kd (gp160). During virus infection of the host cell, gp160 is cleaved by host cell proteases to form gp120 and the integral membrane protein, gp41. The gp41 portion is anchored in the membrane bilayer of virion, while the gp120 segment protrudes into the surrounding environment. gp120 and gp41 are more covalently associated and free gp120 can be released from the surface of virions and infected cells. Furthermore, upon binding to its receptor, CD4, the Env polypeptide undergoes a significant structural rearrangement. After this conformational change the CCR5 co-receptor binding site is exposed. Exposure of the CCR5 binding site, in turn, mediates viral entry into the host cell. See, e.g., Wyatt, R., et al. (1998) Nature 393:705-711; Kwong, P., et al. (1998) Nature 393:648-659. Env appears to be the primary target for inducing a humoral immune response to HIV.
However, it is known that antibodies directed against gp120 do not generally exhibit broad antibody responses against different HIV strains and do not induce production of neutralizing antibodies. See, e.g., Javaherian, K., et al. (1989) Proc. Natl. Acad. Sci. 86:6786-6772; Matsushita, M., et al. (1988) J. Virol. 62:2107-2144; Putney, S., et al. (1986) Science 234:1392-1395; Rushe, J. R., et al. (1988) Proc. Nat. Acad. Sci. USA 85: 3198-3202; Matthews, T. (1986) Proc. Natl. Acad. Sci. USA. 83:9709-9713; Nara, P. L., et al. (1988) J. Virol. 62:2622-2628; Palker, T. J., et al. (1988) Proc. Natl. Acad. Sci. USA. 85:1932-1936).
Furthermore, although neutralizing antibodies are typically generated in the course of HIV infection in humans, these antibodies do not provide permanent antiviral effect may in part be due to the generation of “neutralization escapes” virus mutants and to the general decline in the host immune system associated with pathogenesis. See, e.g., Barre-Sinoussi, F., et al. (1983) Science 220:868-871; Robert-Guroff, M., et al. (1985) Nature (London) 316:72-74; Weis, R., et al. (1985) Nature (London) 316:69-72; Weis, R., et al. (1986) Nature (London) 324:572-575. Nonetheless, it is widely believed that the presence of preexisting neutralizing antibodies upon initial HIV-1 exposure will likely have a protective effect, for instance by attaching to the incoming virions and reducing or preventing their infectivity for target cells and prevent the cell-to-cell spread of virus in tissue culture. See, e.g., Hu et al. (1992) Science 255:456-459; Burton, D., R. and Montefiori, D. (1997) AIDS 11(suppl. A): 587-598; Montefiori and Evans (1999) AIDS Res. Hum. Ret. 15(8):689-698; Bolognesi, D. P., et al. (1994) Ann. Int. Med. 8:603-611; Haynes, B., F., et al. (1996) Science; 271: 324-328.
Several categories of potentially effective neutralizing antibodies have been identified. For example, in most infected individuals, a subset of broadly reactive antibodies that interfere with binding of gp120 and CD4 have been identified. See, e.g., Kang, C.-Y., et al. (1991) Proc. Natl. Acad. Sci. USA. 88:6171-6175; McDougal, J. S., et al. (1986) J. Immunol. 137:2937-2944. Monoclonal antibodies, such as IgG1b12, 2G12 (Mo et al. (1997) J. Virol 71:6869-6874), PA14 (Trkola et al. (2001) J. Virol. 75(2):579-88) and 2F5 also exhibit neutralizing effects. See, also, Trkola et al. (1995) J. Virol. 69:6609-6617; D'Sousa et al (1997) J. Infect. Dis. 175:1062-1075. Other antibodies are believed to bind to the chemokine receptor-binding region after CD4 has bound to Env. See, e.g., Thali et al. (1993) J. Virol. 67:3978-3988). Furthermore, in order to generate antibodies against the CD4 binding site region, which is exposed only upon binding to CD4, several groups have attempted to generate neutralizing antibodies by administering complexes of Env bound to CD4 (e.g., soluble CD4, referred to as “sCD4”) or to CD4 mimetics (e.g., CD4M33). See, e.g., Martin et al. (2003) Nat. Biotechnol. 21(1):71-76.
In addition, WO 04/037847 describes Env-CD4 complexes useful in generating immune responses. Env-CD4 (sCD4) complexes are capable of inducing broadly neutralizing antibodies presumably by targeting conformational epitopes exposed in Env protein upon binding to CD4. However, if sCD4 administered with an adjuvant, the potential for an autoimmune response is of serious concern. In addition, WO 04/037847 describes hybrid Env-CD4 polypeptides.
Despite the above approaches, there remains a need for additional molecules that can elicit an immunological response (e.g., neutralizing and/or protective antibodies) in a subject against multiple HIV strains and subtypes, for example when administered as a vaccine. The present invention solves these and other problems by providing modified Env polypeptides (e.g., gp120) complexed to novel, fusion proteins comprising CD4 mini-proteins or mimics (mimetics) in order to expose epitopes in or near the CD4 binding site.